NK cells play a crucial role in innate defense against pathogens such as murine cytomegalovirus (MCMV). The presence of both activating and inhibitory receptors on the surface of NK cells act collaboratively and in concert with innate cytokines to regulate NK cell functions. Recently, we showed that the killer cell lectin-like receptor G 1 (KLRG 1) is expressed on the most mature NK cells and that infection with MCMV induces an up-regulation of the KLRG1 molecule on the surface of both NK cells and CD8+ T cells. We also demonstrated that KLRG 1 engagement can inhibit NK cell effector functions. In addition, we generated the KLRG1 tetramer and have identified cell lines that are tetramer positive by flow cytometry. Based on these preliminary data, we therefore propose to study several aspects of KLRG 1 functions. In Specific Aim 1, we will use KLRG 1 as a marker of mature NK cells to investigate the NK cell compartmental expansion and contraction phases in response to MCMV infection. We will also examine the consequences of the in vivo blocking of KLRG 1 functions in the context of the MCMV infection of mice in order to determine whether KLRG1 function is to prevent the uncontrolled activation of both NK cells and/or CD8 v T cells. In Specific Aim 2, we will study the biochemical mechanism that dictates the inhibitory properties of the KLRG1 molecule. To do this, transfection/expression analyses of wild-type and mutant KLRG1 molecules will be performed. In Specific Aim 3, we will use a reporter cell line approach, to both confirm the specificity of our tetramer staining and identify cells that express the KLRG1 ligand. In Specific Aim 4, an expression cloning strategy will be developed to clone the KLRG1 ligand. These novel studies will provide insights into the specificity and immunoregulatory role of KLRG1 in particular and NK receptors in general on both NK cells and CD8+ T cells, as well as provide strategies for the regulation of this important category of immune cell responses.